The specific objective of this project is to characterize an unusual transposable element recently discovered in S. cerevisiae. The 341-bp transposable element, sigma, is present in about 30 copies in the haploid genome. In five cases of sigma insertions which have been sequenced, the element is at position -17 or -19 relative to the 5' end of the sequence coding for a mature tRNA and therefore quite close to the point of initiation of transcription. Since these tRNA genes and their flanking sequences are different, it seems likely that the insertion specificity of sigma depends on the conserved internal sequences of the tRNA-coding region. Preliminary studies also indicate that transcription from the tRNA gene can be modified by sigma insertion. Thus a characterization of this system could improve our understanding of transposable elements and provide an opportunity to study the role of flanking DNA in polymerase III gene regulation. The experimental approach to these problems will combine classical yeast suppressor genetics and recombinant DNA technology, including yeast transformation. The experiments are organized around five specific issues: (1) identification of common structural features and tRNA genes associated with sigma in one strain of yeast; (2) search for RNAs homologous to sigma; (3) quantitation of sigma effects on tRNA gene expression; (4) development of an experimental system for detection of sigma transposition and definition of the essential features of the tRNA gene target; and (5) selection of mutations in potential sigma controlling elements. The existence of a transposable element which is clearly associated with a set of genes transcribed by polymerase III and which also affects the activity of those genes gives us an unexpected opportunity to ask very specific questions about the interdependence of two important cellular phenomena--transposition and transcription. The long-range goal of this project is to understand the contribution of transposable elements to transcriptional regulation in eukaryotic cells.